The de novo purine biosynthetic pathway generates purines and their derivatives that act in numerous cellular anabolic and catabolic networks and in cellular information storage. The pathway consists of enzymes that catalyze ten chemical steps and, except for being mono- or multifunctional, are highly conserved from pro- to eukaryotes. Although the enzymes have long been hypothesized to exist in a multienzyme complex, only recently have we been able to demonstrate their clustering in vivo. We propose to investigate the protein components of this cluster, the purinosome, in terms of their stoichiometry and possible inclusion of non-pathway proteins;their protein-protein contacts within the purinosome;the physiological signals that drive purinosome assembly and disassembly;and the advantage of purinosome formation to maintaining purine levels within the cell. Detection and identification of the purinosome associated enzymes and proteins in normal and cancer cell lines will make extensive use of cellular imaging of chimeric fluorescent protein constructs in combination with immunoprecipitation and affinity chromatography to capture purinosome components. Their locus and stoichiometry within the complex, and their putative phosphorylation will be sought by fluorescence resonance energy transfer (FRET), Western blotting and MS/MS spectroscopy methods. The functional advantage of the purinosome to maintain purine levels within cells will be probed by MALDI and secondary ion mass spectrometry (SIMS) and correlated with purinosome cluster density. Our proposed studies focus on the human enzymes and include a significant collaborative effort with the Scripps Molecule Screening Center to discover novel small molecule inhibitors that disrupt purinosome assembly. Many of the individual enzymes, particularly those that utilize folate cofactors, have been biologically validated as chemotherapeutic targets. The proposed studies in the long term have the potential to expand greatly our understanding of how biosynthetic or catabolic pathways may organize intracellularly and the biochemical advantages to the cell of such multienzyme clusters as this approach is extended to other pathways. PUBLIC HEALTH RELEVANCE: The purinosome, consisting of the enzymes responsible for de novo purine biosynthesis, is a novel subcellular organization that can be targeted for chemotherapy. Individual enzymes within the pathway are biologically validated targets. Interference by small molecules with purinosome assembly provides a unique route to new pharmaceuticals.